Polypyrimidine tract-binding protein (PTB) is a nuclear factor that binds to the polypyrimidine tract of premRNA introns, where it is associated with negative regulation of RNA splicing and with exon silencing. We have previously demonstrated that PTB expression is increased during glial cell transformation and that this increase correlates with changes in the RNA splicing of the fibroblast growth factor receptor 1. In this paper we examine the specific cellular distribution of PTB expression in normal brain and in glial and neuronal tumors. Paraffin sections were stained by using a primary monoclonal antibody against PTB. Tissues that were analyzed included normal brain (n = 2) and tumors of various types (low-grade astrocytoma, n = 2; anaplastic astrocytoma, n = 2; glioblastoma, n = 4; medulloblastoma, n = 4; central neurocytoma, n = 2; dysplastic gangliocytoma, n = 1; ganglioglioma, n = 1; paraganglioma, n = 1). In glial cell populations the majority of astrocytes and oligodendrocytes were negative, but occasional positively staining cells were observed. Strongly positive PTB staining was observed in ependymocytes, choroid plexus epithelium, microglia, arachnoid membrane, and adenohypophysis, and weak staining was found in the neurohypophysis. In all cases vascular endothelium and smooth muscle stained strongly. In tumor samples, intense positive nuclear staining was observed in transformed cells of low-grade astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, medulloblastoma, paraganglioma, and the glial population of both ganglioglioma and dysplastic gangliocytoma (the neuronal cells of both were negative). In medulloblastoma, neoplastic neuronal cells were positive, as were other cell lineages. In normal brain, all neuron populations and pineocytes were negative for PTB. We conclude that although glial cells show derepression of PTB expression, a similar mechanism is absent in both nonneoplastic neurons and in most neuronally derived tumor cells. Strong upregulation of PTB expression in tumor cells of glial or primitive neuroectodermal origin suggests involvement of this protein in cellular transformation. Whether PTB affects splicing of RNAs critical to cellular transformation or proliferation is an important question for future research.